The overall goal of this project is to understand molecular mechanisms by which the nuclear bile acid receptor, farnesoid X receptor (FXR), regulates metabolic homeostasis in normal and diseased states. The specific goal of this application is to elucidate the role of post-translational acetylation and deacetylation of FXR mediated by p300 and SIRT1 in FXR function in normal physiology and in pathological conditions. FXR plays a central role in cholesterol/bile acid, fatty acid, lipoprotein, and glucose metabolism by regulating expression of numerous its target genes. Although such important biological roles of FXR are now known, how FXR activity is regulated remains relatively unknown. Transcriptional cofactors, such as p300 acetylase and SIRT1 deacetylase, modulate receptor activity, not only by histone modification of their target gene chromatin, but also by post-translational modification of the receptor itself. In preliminary studies, we found that FXR is acetylated and deacetylated by p300 and SIRT1, respectively. P300 and SIRT1 antagonize each other's activity in the regulation of FXR transactivation. Down-regulation of p300 altered expression of FXR target genes, such that beneficial changes in lipid and glucose metabolic profiles would be expected. FXR acetylation was dynamically regulated under normal physiological states but surprisingly, FXR acetylation levels were substantially elevated in ob/ob mouse liver. Specific lysine residues in FXR were identified as acetylation sites by mass spectrometry and mutation analyses. Interestingly, mutation of these individual sites had different effects on FXR transactivation activity. These intriguing results led us to hypothesize first, that acetylation profoundly modulates FXR activity and is dynamically regulated by p300 and SIRT1 under normal physiological conditions but is highly elevated under metabolic disease and stress conditions and second, that FXR acetylation at different sites may have distinct functional outcomes in normal and disease states by regulating different FXR target genes. To test our hypothesis, we will: 1) determine whether p300 and SIRT1 are critical in vivo cofactors of FXR by down-regulation of these cofactors in cultured cells and in vivo;2) identify acetylated sites in FXR and determine the effects of mutations of these sites on FXR function in vitro, in cultured cells, and in vivo;and 3) determine whether FXR acetylation is dysregulated in pathophysiological conditions and the role of acetylation at specific sites in the disease pathology. Our studies will provide substantial insight into the molecular mechanism of FXR action in vivo and information that may be important for the development of novel therapeutic agents for metabolic disorders, such as fatty liver (liver steatosis), hypercholesterolemia, and diabetes. The bile acid receptor FXR has important biological roles in cholesterol and bile acid homeostasis, triglyceride and lipoprotein metabolism, and glucose regulation in the liver, but how the activity of FXR is regulated remains largely unknown. Our studies to define how FXR acetylation controls FXR activity in health and disease states will provide important information about the mechanisms regulating levels of cholesterol, triglycerides, lipoproteins, and glucose which are abnormal in diseases such as hypercholesterolemia, obesity, and diabetes. The studies may also facilitate the design of therapeutic agents for treating these metabolic disorders.